Reagent composition for fecal occult blood tests

ABSTRACT

Fecal occult blood tests of enhanced sensitivity, specificity, and readability are provided by employing (1) a developer or complete reagent solution that uses as a solvent system a solvent comprising at least about 50% by volume of a solvent for iron protoporphyrins such as dimethyl sulfoxide and/or (2) a multi-chromagen comprised of a mixture of guaiac and ABTS. Test results may be further improved by incorporating hemoprotein solubilizing agents, plant peroxidase inhibitors, iron/copper chelating agents, accelerators and buffers in the developer/reagent.

This application is a divisional of application Ser. No. 869,573, filedJun. 2, 1986, now abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 680,357, filed Dec. 11, 1984, now U.S. Pat. No.4,615,982, and application Ser. No. 502,446, filed Dec. 10, 1985.

DESCRIPTION

Technical Field

This invention is in the field of fecal occult blood tests (FOBT). Moreparticularly it relates to improved FOBT that provide a reducedincidence of false results and/or greater sensitivity and specificityand/or are easier to perform.

Background Art

FOBT are commonly used clinically to detect occult blood loss fromgastrointestinal (GI) lesions. For example, carcinoma of the colon andrectum is the most serious cancer in the U.S. and second only to lungcancer in causing death--approximately 100,000 new cases and 50,000deaths annually. Because colorectal cancer is slowly progressive with along asymtomatic period, it provides an ideal opportunity for earlydetection and successful therapy. Thus, FOBT are a rational attempt atearly diagnosis because the colorectal lesions frequently bleed, androutine noninvasive testing is possible. Similarly, hospitals andphysicians very often utilize FOBT to detect or monitor GI lesionsresulting from disease, injury, surgery, and other causes.

Early FOBT involved shipping entire 24-48 hour fecal collections inpaint cans to central laboratories for testing with an acidified guaiacsolution and hydrogen peroxide. Guaiac is a complex plant extractcontaining the leuco dye, alpha guaiaconic acid. Leuco dyes are oxidizedby hydroperoxides in the presence of catalyst to form a blue color:##STR1## Because hemoglobin is an efficient catalyst (pseudoperoxidase),feces may be tested for occult blood using a leuco dye/hydroperoxidereagent. Nonetheless, the procedure remained very poorly utilizedbecause of the disagreeable nature of the test and physicians werelargely denied this very useful information.

U.S. Pat. No. 3,996,006 describes a FOBT technique that popularized theguaiac-based test for occult blood in feces. It employs a slide having asheet of guaiac-impregnated paper between a front panel and a rear panelwith openings in the panels and pivotal flaps to cover the openings. Afecal specimen is placed on the paper through the opening in the frontpanel and that panel is closed. The rear panel is then opened and ahydrogen peroxide developer is placed on the paper via the opening inthe rear panel. If blood is present in the specimen, the paper will turnblue. A commercial embodiment of this test, called the HEMOCCULT® test,is widely used in hospitals and physicians' offices. Despite thewidespread popularity of the HEMOCCULT® test, recent studies havepointed out serious limitations in its sensitivity and specificity.Applicant believes that the sensitivity limitation is due partly to (1)the fact that hemoglobin in many specimens is degraded to derivativesthat exhibit little or no peroxidative activity, (2) degradation ofperoxidatively active hemoproteins by the hydroperoxide reagent used inthe test and (3) the relative insolubility of the degraded products(i.e., iron protoporphyrins such as heme and hemin) in the reagents usedin the test. Sensitivity limitations, of course, may cause falsenegative results. The specificity limitation is probably due to theresponse of the test to plant peroxidases and/or iron or copper in thespecimens or the environment in which the test is run. Specificitylimitations lead to false positive results.

U.S. Pat. No. 4,333,734 describes a variation in the guaiac-based FOBTthat is intended to reduce the incidence of false positive results dueto the presence of plant peroxidases in the specimen. It includes aperoxidase denaturing agent such as urea or guanidine hydrochloridetogether with a metal chelating agent to sequester calcium and magnesiumions that are essential to peroxidase activity. The denaturant and thechelating agent are formulated with the guaiac.

U.S. Pat. No. 4,071,317 relates to using polar solvents such as dimethylsulfoxide (DMSO) and dimethyl formamide (DMF) to stabilize mixtures oforganic hydroperoxides and leuco dyes that are used in FOBT. The solventis formulated in minor proportions with the hydroperoxide and leuco dye.This solution is applied to a solid matrix and the matrix is dried priorto use in testing.

Several references indicate that monomeric species of ironprotoporphyrins exhibit greater peroxidase activity than dimeric oraggregated species. Biochem J (1970) 117: 741-744; Biochem J (1973) 135:353-359; Biochem J (1976) 153: 279-285; and Biochemistry (1974) 13:4279-4284. Biochem J (1979) 179: 281-289 indicates that hemin occurs inits monomeric form in mixtures of DMSO and water that contain in excessof about 35% (v/v) DMSO.

Biochem J (1968) 108: 131-136 discusses the solubility of nitrogenousligand-alkaline hematin complexes. Biochimica et Biophysica Acta (1977)498: 205-214 describes the use of various water-soluble polymers such aspolyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone, andpolystyrene sulfonate to dissolve aggregates of ferroheme andprotoporphyrin in alkaline aqueous media.

An object of the present invention is to reduce the incidence ofincorrect results (both false positive and false negative) in leucodye-based FOBT. This is achieved by applying the hydroperoxide or boththe hydroperoxide and leuco dye to the specimen in a solution that usesa solvent system that dissolves iron protoporphyrins. Another object isto provide a chromogen that improves test readability and a completeFOBT reagent containing that chromogen.

DISCLOSURE OF THE INVENTION

One aspect of the invention is chromogen for use in a test forperoxidative activity, such as an FOBT, comprising a mixture of guaiacand 2,2'-azinodi-(3-ethylbenzyl)azoline sulfonic acid (ABTS).

Another aspect of the invention is an improvement in a fecal occultblood test method comprising contacting a fecal specimen on a solid testmatrix with a leuco dye and a hydroperoxide, the improvement being thatthe hydroperoxide or the hydroperoxide and the leuco dye are applied tothe fecal specimen in solution in a solvent comprising at least 50% byvolume of a solvent for iron protoporphyrins whereby the ironprotoporphyrins in the specimen are dissolved.

Still another aspect of the invention is a fecal occult blood testmethod comprising contacting a fecal specimen on a solid test matrixwith a leuco dye and a hydroperoxide wherein the leuco dye comprises amixture of guaiac and ABTS.

Still another aspect of the invention is an FOBT reagent comprising ahydroperoxide or a hydroperoxide and a leuco dye in solution in asolvent for iron protoporphyrins selected from the group consisting of:

(a) pyridine;

(b) a mixture of ethanolamine and a co-solvent selected from the groupconsisting of methyl ethyl ketone, tetramethylene sulfone,butyrolactone, glycerol, methanol, tetrahydrofurfuryl alcohol, 2-methoxyethanol, and tetramethyl urea;

(c) a mixture of 2-(diethylamino)ethylamine and a co-solvent selectedfrom the group consisting of methyl ethyl ketone, acetonitrile,tetramethylene sulfone, tetramethyl urea, butyrolactone,tetrahydrofurfuryl alcohol, 2-methoxy ethanol, and methanol;

(d) a mixture of diethanolamine and a co-solvent selected from the groupconsisting of methyl ethyl ketone, acetonitrile, tetramethylene sulfone,butyrolactone, and tetrahydrofurfuryl alcohol; and

(e) 1-methyl-2-pyrrolidinone.

Modes for Carrying Out the Invention

As used herein the term "hemoprotein" is intended to include hemoglobinand derivatives of hemoglobin such as heme and hemin that have theability (particularly in their monomeric form) to catalyze the transferof oxygen from a hydroperoxide to a leuco dye to cause the leuco dye tobe oxidized and thereby produce a detectable response. Such ability issometimes referred to herein as "peroxidative activity".

As used herein the term "leuco dye" is not intended to be limited to aparticular chemical species or genus but is intended to encompassindicators that produce a detectable response, typically a color changethat is visible to the naked eye, when oxidized in the presence of ahemoprotein. Examples of leuco dyes are guaiac, benzidine, o-tolidine,cresol, catechol, 3,3',5,5'-tetramethylbenzidine, p-tolidine,betanaphthol, pyrogallol, o-phenylenediamine, leuco malachite green,3-amino ethylcarbazole, 4-amino antipyrine, phenol, and ABTS.

The various aspects of the invention may be employed separately orcombined to improve FOBT. As indicated one aspect is the use of amixture of guaiac and ABTS as a chromogen in FOBT. Another is applyingto the fecal specimen a developer or complete reagent solution that usesas its solvent system a solvent for iron protoporphyrins.

The chromogen mixture of the invention may be used in various FOBTformats that involve contacting a fecal specimen placed on a solid testmatrix with a leuco dye and a hydroperoxide. In one format the matrix isimpregnated with the mixture (i.e., the matrix carries the mixture indry form) beforehand. In another the matrix is impregnated with one ofthe components of the mixture and the other is applied in solution tothe matrix/specimen either combined with or separately from thehydroperoxide. For long-term stability, it is preferred to keep thechromogen mixture and hydroperoxide separate. For instance, they mightbe packaged in separate containers adapted for simultaneous dispensing,such as a double-barreled syringe with a common outlet nozzle. Anotherformat is to employ the chromogen in solution as part of a completereagent (when long-term stability is not involved). The complete reagentcontains, in addition to the mixture and the solvent, a hydroperoxideand, optionally, other additives such as hemoprotein solubilizingagents, stabilizers, vegetable peroxidase inhibitors, iron chelators,accelerators, and buffers. Such additives may, of course, be impregnatedinto the solid test matrix in the other test formats. Use of a completereagent has the advantages of avoiding the need to impregnate the matrixbeforehand, permitting new test geometries, lower manufacturing costsand use of untreated matrixes, and improving test performance.

In addition to use in FOBT, the chromogen mixture may be usedadvantageously in other common colorimetric tests for the presence ofperoxidatively active molecules other than hemoproteins.

The chromogen mixture of guaiac and ABTS produces a color response thatis greater than the sum of the responses that would be expected from theindividual components. In addition to enhanced color intensity, thecolor is spread more evenly than the observed in current (e.g.,HEMOCCULT® test) FOBT, is more stable (i.e., long-lasting), and is morereproducible. The weight ratio of guaiac to ABTS in the mixture will bein the range of 1:5 to 5:1, preferably approximately 1:1. When thechromogen mixture is formulated as a complete reagent with a peroxideand/or in a solvent for iron protoporphyrins such as DMSO, it ispreferable to include a stabilizing amount of sodium sulfite or otherantioxidant in the formulation. Preferably the sodium sulfite is presentin amounts in excess of that which saturates the solution. Theconcentration of the chromogen mixture in the solution will usually bein the range of 0.5 to 10% by weight.

Similarly, the application to the fecal specimen of a developer/completereagent having a solvent system based on a solvent for ironprotoporphyrins may be used in a variety of FOBT formats. When used as adeveloper, the solution will contain the hydroperoxide and be applied tothe specimen on a leuco dye-impregnated matrix. When used in the form ofa complete reagent, the solution will contain a leuco dye, preferablythe multi-chromogen of the invention, together with the hydroperoxideand, optionally, other additives such as those described above.

The test matrices used in the FOBT methods of the invention may be madefrom a variety of porous materials such as cellulosics (wood, paper),ceramic, glass fibers, natural or synthetic cloth fibers, felt, andsponge. Bibulous filter paper is commonly used and is preferred.

The liquids that are useful as solvents for iron protoporphyrins shouldpreferably have an intermediate to high capacity to dissolve ironprotoporphyrins at a pH in the range of about 6.5 to 7.5. In the presentinstance such capacity was determined by mixing 25 mg crystalline heminwith 1 ml of solvent at ambient temperature, and measuring the amount ofhemin remaining undissolved. Liquids evaluated by this procedure thatexhibited a high capacity for dissolving hemin included aprotic amides,sulfoxides, sulfones, pyridine and mixtures of certain amines and otherorganic solvents.

The amides that were found to be acceptable iron protoporphyrin solventsare of the formula ##STR2## wherein R¹, R², and R³ are the same ordifferent and represent hydrogen, lower alkyl, phenyl or benzyl, withthe provisos that both of R¹ and R² are not hydrogen and R³ may belinked with R¹ or R² to form a 5- or 6-membered heterocycle. The term"lower" as used to modify "alkyl" denotes moieties of 1 to 6 carbonatoms. Examples of such moieties are methyl, ethyl, isopropyl, butyl,and hexyl. Dimethyl formamide and 1-methyl-2-pyrrolidinone are preferredsolvents of this class.

Sulfoxides and sulfones that were found to be acceptable ironprotoporphyrin solvents are of the formula

    R.sup.1 --X--R.sup.2

wherein X is ##STR3## and R¹ and R² are the same or different and arelower alkyl, phenyl, benzyl, or, when X is ##STR4## R¹ and R² may belinked to form a 5 or 6 membered heterocycle (e.g., tetramethylenesulfoxide, pentamethylene sulfoxide).

The mixtures of neutralized amines and organic solvents that were foundto be acceptable iron protoporphyrin solvents include: ethanolamine withglycerol, tetrahydrofurfuryl alcohol, 2-methoxy ethanol, methyl ethylketone, tetramethyl urea, sulfolane (tetramethylene sulfone), orbutyrolactone; 2-(diethylamino)ethylamine with methyl ethyl ketone,acetonitrile, sulfolane, butryolactone, tetrafurfuryl alcohol, 2-methoxyethanol, or methanol and diethanolamine with methyl ethyl ketone,acetonitrile, sulfolane, butyrolactone, or tetrahydrofurfuryl alcohol.Other mixtures of neutralized amines and organic solvents that aresuitable solvents for iron protoporphyrin may be determined empiricallyas described herein. The mixtures are preferably mixed at volume ratiosof 1:1 based on 1M aqueous neutralized amine.

The iron protoporphyrin solvent is also preferably one that wets thetest matrix so that it is capable of transporting (chromatographing)dissolved iron protoporphyrin away from the specimen so that the colorchange, if any, is more easily seen and not obscured by the specimen.

Cosolvents such as water, alkanols (e.g., methanol, ethanol, and otherlower alkanols), pentane, ethylacetate, cyclohexane, and acetone may beincluded with the iron protoporphyrin solvent, but the proportion ofprincipal solvent in the solvent compositions should be maintained aboveabout 50% (v/v), preferably above about 75% (v/v).

Dimethylsulfoxide (DMSO) is a particularly preferred iron protoporphyrinsolvent. The use of DMSO as the solvent in the developer/completereagent provides several advantages. Firstly, it is an excellent solventfor hemoproteins, including iron protoporphyrins. Secondly, it convertsheme and hemin dimers and aggregates into the monomeric forms. And,thirdly, it inactivates peroxidases that may be present in the fecalspecimen and inhibits them from catalyzing the oxidation of theindicator and producing a false positive result.

Hemoprotein solubilizing agents that may be optionally included in thesolution or test matrix include detergents and water-soluble polymers.Detergents that have suitable hydrophilic-hydrophobic balance tosolubilize hemoproteins are suitable. Such detergents include theTRITON® detergents (polyoxyethylene alkylphenols), detergents from theseries alkyltrimethylammonium bromides, like cetylalkyltrimethylammonium bromide (CTAB) or p-toluene sulfonic acid saltsof alkyltrimethylammonium bromide detergents, and C₁₀ to C₁₄ alkalimetal salts of fatty acids or alkali metal alkyl sulfates. Examples ofsuitable detergents are sodium dodecyl sulfate (SDS), sodium dodecylsulfonate, sodium decyl sulfate, sodium tetradecyl sulfate, sodiumtridecyl sulfonate, sodium myristate, sodium caprate, sodium dodecylN-sarcosinate, and sodium tetradecyl N-sacrosinate. The water-solublepolymers that may be used to solubilize hemoproteins includepoly(ethylene oxide), poly(vinyl alcohol), poly(vinyl pyrrolidone),poly(vinyl pyridine), and poly(styrene sulfonate). These solubilizingagents not only solubilize hemoproteins, but are believed to convertperoxidatively inactive hemoprotein dimers or aggregates intoperoxidatively active monomeric species.

Nitrogenous ligands that stabilize iron protoporphyrins and enhancetheir peroxidative activity may also be incorporated in the completereagent or test matrix. Examples of such ligands are pyridine,histidine, caffeine, imidazole, and imidazole derivatives.

Quinoline and quinoline derivatives may be included in thedeveloper/complete reagent to accelerate the color formation. Apreferred quinoline derivative is7-chloro-4-(4-diethylamino-1-methyl(butylamino)quinoline.

Other additives such as vegetable peroxidase inhibitors and ironchelators (e.g., ethylenediamine tetraacetic acid, N, N, N', N'-diaminocyclohexane tetraacetic acid, citric acid, tartaric acid,nitrilotriacetic acid, diethylenetriamine-pentaacetic acid,N,N'-bishydroxyethyl glycine, ethyleneglycolbis(2-aminoethylether)tetraacetic acid,N-hydroxyethylethylenediaminetetraacetic acid) may be incorporated intothe test matrix to further reduce the likelihood of false test results.Buffers are added to maintain a suitable pH range for oxidizing theleuco dye. The particular buffer (pH range) will depend on the leuco dyethat is used. The pH will usually be between about 3 and about 9. By wayof example, guaiac oxidation is buffered at pH 6-7.5 (phosphate buffer),3,3',5,5'-tetramethylbenzidine oxidation is buffered at a pH of about 4(acetate buffer), and ABTS is buffered at a pH of about 9-9.5 (glycinebuffer). For the chromogen mixture of the invention the pH should be inthe range of 5 to 10.

Hydrogen peroxide or organic hydroperoxides such as cumenehydroperoxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide,and 2,5-dimethylhexane hydroperoxide may be used in the developer orcomplete reagent. It is preferred to use an organic hydroperoxide sinceorganic hydroperoxides are less likely to (a) produce false positiveresults in FOBT in which vegetable peroxidases are present in the fecalspecimen and (b) destroy the peroxidase activity of the hemoprotein. Theconcentration of hydroperoxide in the developer/complete reagent willusually be in the range of 0.05 to 10% by volume, more usually 0.5 to 5%by volume.

The FOBT materials and procedures of the invention and their advantagesover previous materials and procedures are illustrated by the followingexamples. These examples are not intended to limit the invention in anymanner.

EXAMPLE 1

Tests were carried out to compare the specificity and sensitivityachieved in FOBT with a developer composition consisting of (5% (v/v)cumene hydroperoxide (CHP) in DMSO) versus that achieved with aconventional FOBT developer (3-6% (v/v) hydrogen peroxide inethanol/water).

Serial dilutions of hemin (Hm, 0.1 mg/ml in 1 mM ammonium hydroxide),hemoglobin (Hb, 1.0 mg/ml), and horseradish peroxidase (HRPO, 20 U/ml)in water were prepared. Two μl of these dilutions were applied toHEMOCCULT® slides and the spots were allowed to dry. Five μl ofdeveloper composition was applied to each spot. After one minute thepresence or absence of blue color on the slide was noted (+++=intensecolor; ++=strong color; +=clearly visible color; ±=barely perceptiblecolor; -=no detectable color). The results of these tests are shown inTable 1 below.

                  TABLE 1                                                         ______________________________________                                        Hm              Hb           HRPO                                             Dilution                                                                              H.sub.2 O.sub.2                                                                       CHP     H.sub.2 O.sub.2                                                                      CHP   H.sub.2 O.sub.2                                                                     CHP                                ______________________________________                                        0       +++     +++     +++    +++   +++   +++                                2       +++     +++     +++    +++   +++   +                                  4       +++     +++     +++    +++   +++   ±                               8       ++      ++      +++    ++    +++   -                                  16      ±    +       +      ++    +++   -                                  32      -       ±    ±   +     ++    -                                  64      -       -       ±   ±  ++    -                                  128     -       -       -      ±  +     -                                  256     -       -       -      -     ±  -                                  512     -       -       -      -     ±  -                                  ______________________________________                                    

As indicated by the results of Table 1, the DMSO-based developer wasmore sensitive than the conventional developer with hemoglobin and heminand much less sensitive than the conventional developer with horseradishperoxidase. The invention developer thus improves the sensitivity andspecificity of the test. Sensitivity can be improved by modifying thedeveloper as needed.

EXAMPLE 2

Hemin degradation tests were carried out using conventional FOBTdeveloper and DMSO-based FOBT developer compositions of the presentinvention as follows.

Hemin was dissolved in a 1:10 (v/v) mixture of DMSO and water. Two μl ofthis solution was spotted onto filter paper and the paper was air dried.Spotted areas were then treated with 5 μl of one of the followingdevelopers: (a) CHP, 5%, in DMSO; (b) t-butyl hydroperoxide, 5%, inDMSO; (c) H₂ O₂, 5% in water; (d) HEMOCCULT® developer (H₂ O₂, 5%, inethanol/water); (e) DMSO alone; (f) water alone; and (g) 75% ethanol,25% water. The developer-treated areas were then air dried and 10 μl ofguaiac solution (30 g/1200 ml isopropanol) was applied to each spotfollowed by air drying. Each spot was then treated with 15 μl HEMOCCULT®developer and color change was observed. Visible blue color was noted oneach spot except those treated with (c) and (d). These results indicatethat organic hydroperoxides in DMSO do not effect adversely thecatalytic activity of hemin, whereas conventional FOBT developer doeshave an adverse effect on hemin activity in FOBT. Similar results wereobserved using native and denatured hemoglobin instead of hemin.

EXAMPLE 3

Tests were conducted using developer compositions consisting of CHP at5% in DMSO with various cosolvents. The procedure of Example 1 was usedexcept that the highest clearly detectable dilution of catalyst is notedrather than color intensity. Conventional developer and CHP in othersolvents were run for comparison. The results are reported in Table 2below.

                  TABLE 2                                                         ______________________________________                                                             Highest                                                                       Detectable Dilution                                      Oxidant     Solvent        Hm     Hb   HRPO                                   ______________________________________                                        Hydrogen peroxide                                                                         ˜75% ethanol                                                                           5      5    10                                     CHP         100% DMSO      6      ND   4                                      CHP         75% DMS0       5      ND   5                                                  25% Pentane                                                       CHP         50% DMSO       5      ND   6                                                  50% Pentane                                                       CHP         75% DMSO       5      ND   5                                                  25% ethyl acetate                                                 CHP         75% DMSO       5      ND   5                                                  25% Cyclohexane                                                   CHP         50% DMSO       5      ND   5                                                  50% Cyclohexane                                                   CHP         Dimethyl Formamide                                                                           7      8    8                                      CHP         Acetonitrile   7      ND   9                                      CHP         90% DMSO       4      4    3                                                  10% Acetone                                                       CHP         75% DMSO       4      5    3                                                  25% Acetone                                                       CHP         50% DMSO       4      5    5                                                  50% Acetone                                                       CHP         100% EtOH      4      6    7                                      CHP         100% MeOH      5      8    7                                      ______________________________________                                         ND = not determined                                                      

The results of Table 2 show that CHP developer with DMSO at >50% showreduced sensitivity to HRPO while retaining sensitivity for hemin. Thebest results occur using DMSO at 75% and above.

EXAMPLE 4

Tests were carried out to assess the effect of the developer compositionon the mobility of hemin spotted on filter paper. This is an indirectmeasure of the solubility of hemin in the developer.

Whatman filter paper was soaked with 10 μl of a 1 mg/ml hemin solutionin 1 mM aqueous ammonium hydroxide. After drying, a small section of thehemin-treated paper was placed on a HEMOCCULT® slide. Ten μl ofdeveloper compositions comprising 5% CHP in DMSO/water solventcontaining 100%, 95%, 90%, 80%, 75%, 50%, and 25% (v/v) DMSO were placedon the hemin spots and blue color formation was observed. At DMSOconcentrations above about 75% blue color was noted in the entire slidearea wetted by the developer. At lower DMSO concentrations color wasobserved only directly under the hemin-treated filter paper. A similartest with HEMOCCULT® developer produced no color.

Similar tests using hemoglobin instead of hemin gave similar results.

EXAMPLE 5

This example illustrates the effect of hemin solubility on thereadability of FOBT test results.

A normal fecal specimen which tested negative for fecal occult bloodwith the HEMOCCULT® FOBT and with an FOBT reagent consisting of 2%guaiac, 1% ABTS, 5% cumene hydroperoxide, and 25 mg/ml7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline in 75% DMSO/25%water was spiked with a DMSO solution of hemin sufficient to produce afinal hemin concentration of 240 μg/g feces. This level of hemin isequivalent in iron protoporphyrin to 6 mg hemoglobin/g feces. Thinsmears of the spiked fecal specimen were added to standard, untreatedfilter paper and allowed to air dry.

A second FOBT formulation was prepared in tetramethyl urea (2% guaiac,1% ABTS and 5% cumene hydroperoxide). Fifty microliters of the new FOBTformulation in tetramethyl urea was added to a smear of the spiked fecalspecimen on filter paper. No blue color was eluted from the specimenonto the surrounding filter paper. The specimen became somewhat darkerin appearance, but it was extremely difficult to decide if a blue colorwas present because of the dark fecal background.

When a drop of DMSO was added to the spiked fecal specimen which hadbeen treated with the second FOBT formulation, a dark blue color waseluted immediately from the fecal specimen onto the surrounding paper.Similarly, when a drop of aqueous 1M ethanolamine, pH 7, was added to aspiked fecal specimen which had been treated with the second FOBTformulation, a dark blue color was immediately eluted from the fecalspecimen onto the surrounding paper. DMSO is a good heme solvent, and a1:1 mixture of tetramethyl urea and aqueous 1M ethanolamine, pH 7, is agood solvent for hemin even though neither tetramethyl urea nor aqueous1M ethanolamine alone is a good hemin solvent.

These observations indicate that a good solvent for iron protoporphyrinsis extremely important in eluting the color produced in a positive FOBTfrom a fecal specimen containing iron protoporphyrins. Moreover, thelonger the fecal specimen is allowed to dry, the more critical the roleof iron protoporphyrin solvent in this regard. In screening programs forcolorectal cancer, it is common for fecal smears to be stored for up to14 days prior to performing the test for fecal occult blood.

EXAMPLE 6

The effect of hemoglobin denaturation on FOBT sensitivity using aDMSO-based developer composition of the invention and HEMOCCULT®developer was determined as follows.

Hemoglobin was denatured by treatment with (a) hydrochloric acid, (b)enzymes or (c) heat as follows:

(a) Acid

Dilute (0.1M) hydrochloric acid was added to a solution of hemoglobin inwater (5 mg/ml) until the pH of the solution was 1.0. The solution wasincubated for 1 hr at 37° C. and the pH adjusted to 7.4 with 0.1N sodiumhydroxide. The resulting material was homogenized to produce a finelydispersed suspension and incubated at 37° C. for 48 hr.

(b) Enzymes

Solid trypsin (1 mg) and chymotrypsin (1 mg) were added to a solution ofhemoglobin in water (5 mg/ml) and incubated for 48 hr at 37° C. Theresulting mixture was homogenized to produce a smooth suspension.

(c) Heat

A solution of hemoglobin in water (5 mg/ml) was heated to 75° C. for 10min and incubated at 37° C. for 48 hr. The resulting mixture washomogenized to produce a smooth suspension.

Standard HEMOCCULT® slides were spotted with 2 μl of an aqueous solution(5 mg/ml) of the denatured hemoglobins or native hemoglobin dilutedserially 1:2 with water and allowed to dry. The slides were then treatedwith developer composition (designated CHP in Table 3 below) consistingof 5% CHP in DMSO/water. Comparison slides were treated with HEMOCCULT®developer (designated HO in Table 3 below). Color intensities wereobserved as above. Table 3 reports the results of these tests.(+5=extremely intense color; +1=slight color; -=no color; ±=barelydetectable.)

                                      TABLE 3                                     __________________________________________________________________________                   HEMOGLOBIN TREATMENT                                                   NATIVE ACID    ENZYME HEAT                                            DILUTION                                                                              HO  CHP                                                                              HO  CHP HO  CHP                                                                              HO  CHP                                         __________________________________________________________________________     2      5   5  3   3   5   5  5   5                                            4      5   5  3   3   4   5  5   5                                            8      4   5  2   2   4   4  4   5                                            16     4   4  1   2   3   4  3   4                                            32     3   4  +/- 1   2   3  2   3                                            64     3   3  +/- 1   2   3  1   3                                           128     2   3  --  1   1   2  1   2                                           256     2   2  --  1   +/- 2  +/- 2                                           512     1   2  --  1   --  1  --  1                                           1024    +/- 2  --  +/- --  1  --  1                                           __________________________________________________________________________

The data reported in Table 3 indicate that the sensitivity of the FOBTusing the DMSO-based developer is not affected by hemoglobindenaturation as much as FOBT using conventional developers. This isparticularly significant since such denaturation is likely to occur tohemoglobin in the GI tract.

EXAMPLE 7

This example illustrates the enhanced readability of the multi-chromagenFOBT reagent as regards color intensity and diffusiveness.

The following reagents were prepared:

A. HEMOCCULT® developer (6% hydrogen peroxide in alcohol);

B. 5% cumene hydroperoxide and 2% guaiac in 75% DMSO, 25% water; and

C. 5% cumene hydroperoxide, 2% guaiac, and 25 mg/ml ABTS in 75% DMSO,25% water containing 100 mg solid sodium sulfite.

Standard HEMOCCULT® slides were smeared with untreated fecal specimens,or with specimens which had been spiked with hemin or hemoglobin (135 or270 μg hemin/g feces, or with 5.7 mg hemoglobin/g feces). After thesmears had dried, the appropriate reagent was added, and the appearanceof the test noted at 1 min. The results are reported in Table 4.

                  TABLE 4                                                         ______________________________________                                                   Hydrogen   CHP +    ABTS + CHP +                                   Fecal Specimen                                                                           Peroxide   Guaiac   Guaiac                                         ______________________________________                                        1.  Untreated  negative   negative                                                                             negative                                     2.  Hemin spiked                                                                             negative   +1     +5                                               (135 μg/g)         (hollow                                                                              (solid circle)                                                         center)                                             3.  Hemin spiked                                                                             negative   +3     +10                                              (270 μg/g)         (hollow                                                                              (solid circle)                                                         center)                                             4.  Hemoglobin +1         +3     +10                                              spiked                       (solid circle)                                   (2.9 mg/ml)                                                               ______________________________________                                         negative = no color produced.                                                 +1 = very faint blue color.                                                   +10 = extremely intense blue color.                                           hollow center = blue ring with color at edge of solvent front, and no         color in center.                                                              solid circle = even blue color throughout the area covered by the solvent                                                                              

EXAMPLE 8

This example shows the relative sensitivity and readability of FOBTsusing the multi-chromogen of the invention and reagents containing onlyABTS or guaiac.

The following chromogen compositions were used:

A. 2% guaiac and 5% cumene hydroperoxide in 75% DMSO, 25% water.

B. ABTS (10 mg/ml) and 5% cumene hydroperoxide in 75% DMSO, 25% water.

C. 1% guaiac, 5% cumene hydroperoxide, and ABTS (5 mg/ml) in 75% DMSO,25% water.

A solution of hemin in DMSO (10 mg/ml) was diluted with DMSO in serialfashion 1:2 and the dilutions were spotted (2 μl) onto standard Whatman#1 filter paper. Five μl of reagent was added and color formation notedat 60 sec. The results of these tests are reported in Table 5.

                  TABLE 5                                                         ______________________________________                                        Color Formation                                                               Hemin Concentration                                                           (mg/ml)      Reagent A  Reagent B Reagent C                                   ______________________________________                                        1.00         +          -         +                                           0.50         +          -         +                                           0.25         +          -         +                                           0.13         +          -         +                                           0.07         +          -         +                                           0.04         +          -         +                                           0.02         +          -         +                                           0.01         +/-        -         +                                           0.005        +/-        -         +/-                                         0.0025       -          -         +/-                                         ______________________________________                                    

Color Characteristics:

Reagent A=Blue ring at solvent front, but hollow, colorless center.

Reagent B=No color formed on standard filter paper.

Reagent C=Very intense blue color formed; color evenly distributedthroughout the entire solvent area.

EXAMPLE 9

This example shows the chromatographic advantages of the multi-chromogenreagent relative to a single chromogen reagent.

The following reagents were used:

A. Reagent A-2% guaiac and 5% cumene hydroperoxide in DMSO.

B. Reagent B-Multi-chromogen reagent (2% guaiac, 5% cumenehydroperoxide, and 25 mg/ml ABTS in DMSO over 100 mg solid sodiumsulfite).

Two μl of a hemin suspension in water (1 mg/ml) was spotted ontostandard Whatman #4 filter paper. After the water had evaporated,varying volumes of Reagent A or Reagent B were added, and the appearanceof the blue color noted. Table 6 reports the results of these tests.

                  TABLE 6                                                         ______________________________________                                                  Appearance of Spot                                                  Reagent Volume                                                                            REAGENT A     REAGENT B                                           ______________________________________                                         5 μl    very slightly full circle, intense                                            disperse      color                                               10 μl    slightly disperse,                                                                          full circle, intense                                            hollow center color                                               15 μl    disperse circle,                                                                            full circle, intense                                            hollow center color                                               20 μl    very disperse intense color, very                                             circle, hollow                                                                              slightly disperse                                               center        center                                              ______________________________________                                    

Color Characteristics:

full circle=Intense blue circle with color evenly distributed throughoutthe entire area covered by the solvent.

very slightly disperse circle=Blue circle, but small, faint, colorlessarea at center of circle.

slightly disperse=Blue circle, approximately 20% of central areacolorless.

disperse circle=Blue ring, approximately 50% of central area colorless.

very disperse circle=blue ring with >80% of the central area colorless;color is primarily localized at the solvent front.

EXAMPLE 10

This example illustrates enhanced color intensity and uniformityobtained using the multi-chromogen reagent.

The following reagents were used:

A. 2% guaiac and 5% cumene hydroperoxide in DMSO.

B. 2% guaiac and 5% cumene hydroperoxide and ABTS (25 mg/ml) in DMSOcontaining 100 mg solid sodium sulfite.

Two μl hemin solution (0.05 mg/ml) in DMSO was spotted onto untreatedWhatman #1 filter paper. Ten μl reagent A or B was added and theappearance noted in 1-3 min. Each test was run in triplicate. Table 7reports the results of the tests.

                  TABLE 7                                                         ______________________________________                                        REAGENT A         REAGENT B                                                   Intensity                                                                              Appearance   Intensity                                                                              Appearance                                     ______________________________________                                        1.  +3       Blue ring with                                                                             +10    Solid blue circle                                         hollow center       intensely blue                               2.  +3       Blue ring with                                                                             +10    Solid blue circle                                         hollow center       intensely blue                               3.  +3       Blue ring with                                                                             +10    Solid blue circle                                         hollow center       intensely blue                               ______________________________________                                    

Color Characteristics:

Blue ring with hollow center=Blue color at edge of solvent front, but nocolor in center.

Solid blue circle=Even, blue color throughout the area covered by thesolvent.

    ______________________________________                                                +1  = very faint blue                                                         +10 = extremely intense blue                                          ______________________________________                                    

EXAMPLE 11

This example provides a comparison of color intensity, stability andchromatography in tests using the HEMOCCULT® reagents and the multiplechromogen of the invention, with hemin as catalyst.

The following reagents were used:

A. HEMOCCULT® developer (6% hydrogen peroxide in alcohol).

B. Multi-chromogen reagent (1% guaiac, 5% cumene hydroperoxide, and ABTS(25 mg/ml) in DMSO).

Two μl of a suspension of hemin in water (0.25 mg/ml) was added tostandard HEMOCCULT® slides, and the spots allowed to dry. Five μl ofeach reagent was added to the spots, and color formation noted at 30sec, 60 sec, 3 min, and 5 min. Table 8 reports the results of thesetests.

                  TABLE 8                                                         ______________________________________                                        COLOR FORMATION                                                                                   MULTIPLE                                                  HEMOCCULT           CHOMOGEN                                                  DEVELOPER           REAGENT                                                   Time  Intensity                                                                              Appearance   Intensity                                                                            Appearance                                 ______________________________________                                        30 sec                                                                              +2       diffuse uneven                                                                             +8     intense full                                              color               circle color                               60 sec                                                                              +1       diffuse, uneven                                                                            +10    intense full                                              color               circle color                               3 min neg      no color     +7     intense full                                                                  circle color                               5 min neg      no color     +7     intense full                                                                  circle color                               ______________________________________                                    

Color Characteristics:

Reagent A=Weak, diffuse color with streaking.

Reagent B=Extremely intense blue color. Color is evenly distributedthroughout the area covered by the solvent.

EXAMPLE 12

This example reports the sensitivity of complete multiple-chromogen FOBTto plant peroxidases relative to other reagents.

The following reagents were used:

A. Horseradish Peroxidase, 1,000 Units/ml.

B. HEMOCCULT® developer (6% hydrogen peroxide in alcohol).

C. 5% cumene hydroperoxide in DMSO.

D. 5% cumene hydroperoxide in DMSO containing 2% guaiac.

E. 5% cumene hydroperoxide in DMSO containing 2% guaiac and ABTS (25mg/ml) stored over solid sodium sulfite.

The horseradish peroxidase was diluted in serial fashion with 1:2 withdistilled water. Two μl of the dilutions were spotted in quadruplicateon standard HEMOCCULT® slides. The spots were allowed to dry, and 5 μlof the appropriate reagent added. Color was determined at 60 sec. Theresults of these tests are reported in Table 9.

                  TABLE 9                                                         ______________________________________                                        COLOR FORMATION                                                                       REA-      REA-      REA-     REA-                                     (Units/ml)                                                                            GENT B    GENT C    GENT D   GENT E                                   ______________________________________                                        2.00    +         +         +        +                                        1.00    +         +         +        +                                        0.50    +         +         +        +                                        0.25    +         +         +        -                                        0.13    +         +/-       +        -                                        0.07    +         -         +        -                                        0.04    +         -         +/-      -                                        0.02    +         -         +/-      -                                        0.01    +         -         -        -                                        0.005   +         -         -        -                                        0.0025  +/-       -         -        -                                        0.0013  +/-       -         -        -                                        0.0007  +/-       -         -        -                                        0.0004  +/-       -         -        -                                        0.0002  -         -         -        -                                        ______________________________________                                    

EXAMPLE 13

This example shows the effect of sulfite on the multiple-chromogenreagent stability.

Multiple component reagent containing 5% cumene hydroperoxide and 10.1mg/ml ABTS in DMSO was made by adding guaiac or solid sodium sulfite asindicated in Table 10 below. Sodium sulfite was present either as excesssolid, or as a saturated solution in DMSO from which all crystals wereremoved by filtration.

Procedure:

Each reagent was tested for its ability to detect 2 μl of a heminsolution (1 mg/ml) in DMSO on standard Whatman filter paper #1. Ten μlof reagent were used. The results are reported in Table 10.

                  TABLE 10                                                        ______________________________________                                                              COLOR                                                   REAGENT COMPOSITION   FORMATION                                               ABTS (%)                                                                              Guaiac (%)                                                                              Sulfite Added                                                                             Day One                                                                              Day 120                                  ______________________________________                                        1.0     2.0       none        +      -                                        1.0     2.0       saturated (no                                                                             +      -                                                          excess solid)                                               1.0     2.0       excess solid                                                                              +      +                                        1.0     none      none        +/-    -                                        1.0     none      excess solid                                                                              +/-    -                                        ______________________________________                                    

EXAMPLE 14

This example illustrates other iron protoporphyrin solvents comprising amixture of ethanolamine, 2-(diethylamino)ethylamine, or diethanolaminewith various organic solvents which, by themselves are not good ironprotoporphyrin solvents.

Twenty-five mg crystalline hemin was added to a test tube. One ml ofamine-containing solvent was added to the tube and the tube wasvortexed. Amines used were in 1M aqueous solutions at pH 7. Amines weremixed with co-solvents at a volume ratio of 1:1. The solubility of thehemin in the solvent was evaluated using the following scale.0=non-miscible, 1=slight solubility, 2 to 4=intermediate solubility, and5=complete solubility. Solvent systems having readings of 4 or 5 wereconsidered acceptable FOBT solvents. Table 11 below reports the resultsof these tests.

                  TABLE 11                                                        ______________________________________                                                   Hemin Solubility                                                                                 2-(diethyl-                                                                            Die-                                                        Ethanol- amino)ethyl-                                                                           thanol                                 Co-solvent   None    amine    amine    mine                                   ______________________________________                                        None         x       1        2        1                                      Methyl ethyl 1       5        5        5                                      ketone                                                                        Acetonitrile 0       0        5        5                                      Tetramethylene                                                                             1       5        5        5                                      sulfone                                                                       Butyrolactone                                                                              2       5        5        4                                      Tetrahydrofurfuryl                                                                         1       5        5        5                                      alcohol                                                                       2-Methoxy ethanol                                                                          1       5        5        5                                      Methanol     1       3        5        2                                      Tetramethyl urea                                                                           2       5        x        x                                      Glycerol/H.sub.2 0 1:1                                                                     0       4        x        x                                      v/v                                                                           ______________________________________                                    

Modifications as the above modes for carrying out the invention that areobvious to those of skill in the fields of medicine, biochemistry and,in particular FOBT, are intended to be within the scope of the followingclaims.

I claim:
 1. A complete reagent composition for use in a fecal occultblood test comprising a solution of a leuco dye and a hydroperoxide in asolvent containing at least about 50% by volume of a solvent for ironprotoporphyrins, wherein said solvent for iron protoporphyrins is amixture of a neutralized amine and an organic co-solvent, wherein theneutralized amine is selected from the group consisting of ethanolamine,2-(diethylamino)ethylamine, and diethanolamine.
 2. The composition ofclaim 1, wherein the pH of the solution is in the range of about 6.5 toabout 7.5.
 3. The composition of claim 1, wherein the hydroperoxide isan organic hydroperoxide.
 4. The composition of claim 3, wherein theorganic hydroperoxide is cumene hydroperoxide.
 5. The composition ofclaim 1, wherein the leuco dye comprises a mixture of guaiac and2,2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
 6. Thecomposition of claim 5, wherein the weight ratio of guaiac to ABTS is inthe range of 1:5 to 5:1.
 7. A complete reagent composition for use in afecal occult blood test comprising a solution of a leuco dye indicatorand a hydroperoxide in a solvent comprising at least about 50% by volumeof a solvent for iron protoporphyrins, wherein said solvent for ironprotoporphyrins is pyridine, and wherein the pH of the solution is inthe range of about 6.5 to about 7.5.
 8. The composition of claim 7,wherein the hydroperoxide is an organic hydroperoxide.
 9. Thecomposition of claim 8, wherein the organic hydroperoxide is cumenehydroperoxide.
 10. The composition of claim 7, wherein the leuco dyecomprises a mixture of guaiac and 2,2'-azino-di-(3-ethylbenzyl) azolinesulfonic acid (ABTS).
 11. The composition of claim 10, wherein theweight ratio of guaiac to ABTS is in the range of 1:5 to 5:1.
 12. Acomplete reagent composition for use in a fecal occult blood testcomprising a solution of a leuco dye indicator and a hydroperoxide in asolvent comprising at least about 50% by volume of a solvent for ironprotoporphyrins, wherein said solvent for iron protoporphyrins isethanolamine and a co-solvent selected from the group consisting ofglycerol, methyl ethyl ketone, tetramethylene sulfone, butyrolactone,tetrahydrofurfuryl alcohol, 2-methoxy ethanol and tetramethyl urea, andwherein the pH of the solution is in the range of about 6.5 to about7.5.
 13. The composition of claim 12, wherein the ethanolamine isapproximately 1M aqueous ethanolamine and the volume ratio of theaqueous ethanolamine to co-solvent is approximately 1:1.
 14. Thecomposition of claim 12, wherein the hydroperoxide is an organichydroperoxide.
 15. The composition of claim 14, wherein the organichydroperoxide is cumene hydroperoxide.
 16. The composition of claim 12,wherein the leuco dye comprises a mixture of guaiac and2,2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
 17. Thecomposition of claim 16, wherein the weight ratio of guaiac to ABTS isin the range of 1:5 to 5:1.
 18. A complete reagent composition for usein a fecal occult blood test comprising a solution of a leuco dyeindicator and a hydroperoxide in a solvent comprising at least about 50%by volume of a solvent for iron protoporphyrins, wherein said solventfor iron protoporphyrins is 2-(diethylamino)ethylamine and a co-solventselected from the group consisting of methyl ethyl ketone, acetonitrile,tetramethylene sulfone, tetramethyl urea, butyrolactone,tetrahydrofurfuryl alcohol, 2-methoxy ethanol, and methanol, and whereinthe pH of the solution is in the range of about 6.5 to about 7.5. 19.The composition of claim 18, wherein the 2-(diethylamino)ethylamine isapproximately 1M aqueous 2-(diethylamino)ethylamine and the volume ratioof the aqueous 2-(diethylamino)ethylamine to co-solvent is approximately1:1.
 20. The composition of claim 18, wherein the hydroperoxide is anorganic hydroperoxide.
 21. The composition of claim 20, wherein theorganic hydroperoxide is cumene hydroperoxide.
 22. The composition ofclaim 18, wherein the leuco dye comprises a mixture of guaiac and2,2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
 23. Thecomposition of claim 22, wherein the weight ratio of guaiac to ABTS isin the range of 1:5 to 5:1.
 24. A complete reagent composition for usein a fecal occult blood test comprising a solution of a leuco dyeindicator and a hydroperoxide in a solvent comprising at least about 50%by volume of a solvent for iron protoporphyrins, wherein said solventfor iron protoporphyrins is diethanolamine and a co-solvent selectedfrom the group consisting of methyl ethyl ketone, acetonitrile,tetramethylene sulfone, butyrolactone, and tetrahydrofurfuryl alcohol,and wherein the pH of the solution is in the range of about 6.5 to about7.5.
 25. The composition of claim 24, wherein the diethanolamine isapproximately 1M aqueous diethanolamine and the volume ratio of theaqueous diethanolamine to co-solvent is approximately 1:1.
 26. Thecomposition of claim 24, wherein the hydroperoxide is an organichydroperoxide.
 27. The composition of claim 26, wherein the organichydroperoxide is cumene hydroperoxide.
 28. The composition of claim 24,wherein the leuco dye comprises a mixture of guaiac and2,2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
 29. Thecomposition of claim 28, wherein the weight ratio of guaiac to ABTS isin the range of 1:5 to 5:1.
 30. A complete reagent composition for usein a fecal occult blood test comprising a solution of a leuco dyeindicator and a hydroperoxide in a solvent comprising at least about 50%by volume of a solvent for iron protoporphyrins, wherein said solventfor iron protoporphyrins is 1-methyl-2-pyrrolidinone, and wherein the pHof the solution is in the range of about 6.5 to about 7.5.
 31. Thecomposition of claim 30, wherein the hydroperoxide is an organichydroperoxide.
 32. The composition of claim 31, wherein the organichydroperoxide is cumene hydroperoxide.
 33. The composition of claim 30,wherein the leuco dye comprises a mixture of guaiac and2,2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
 34. Thecomposition of claim 33, wherein the weight ratio of guaiac to ABTS isin the range of 1:5 to 5:1.